Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. Antibodies are made up of two distinct regions, referred to as the variable (Fv) and constant (Fc) regions. The light and heavy chain Fv regions contain the antigen binding determinants of the molecule and are responsible for binding the target antigen. The Fc regions define the class (or isotype) of antibody (IgG for example) and are responsible for binding a number of natural proteins to elicit important biochemical events.
The Fc region of an antibody interacts with a number of ligands including Fc receptors and other ligands, imparting an array of important functional capabilities referred to as effector functions. An important family of Fc receptors for the IgG class are the Fc gamma receptors. These receptors mediate communication between antibodies and the cellular atm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). An important type of Fc gamma receptors for the IgG class is the neonatal Fc Receptor (FcRn). FcRn is a heterodimer, which comprises β2-microglobulin and a membrane-anchored α chain that is related to the β chain of major histocompatibility complex class I molecules (Simister et al., 1989, Nature 337, 184-187; Burmeister et al., 1994, Nature 372, 336-343). FcRn recycles IgGs within endothelial cells and rescues them from a degradative pathway (Brambell et al., 1964, Nature 203, 1352-1354; Junghans et al., 1996, Proc. Natl. Acad Sci., 93, 5512-5516; Ghetie and Ward, 2000, Annu. Rev. Immunol., 18, 739-766; Roopenian & Akilesh, 2007, Nat. Rev. Immunol., 7, 715-725). The most notable feature of the interaction between IgG Fc and FcRn is its pH dependency: the Fc portion of IgGs binds FcRn with a high affinity at pH 6.0 and is released at pH 7.2 (Rodewald, 1976, J. Cell Biol., 71:666-669; Raghavan et al., 1995, Biochemistry, 34:14649-14657). This crucial characterstic is intricately linked to the IgG salvage mechanism, which involves recycling FcRn bound IgGs from within acidic lysosomes back to general circulation (Ghetie and Ward, 2000, Annu. Rev. Immunol., 18, 739-766). As result, recycled IgGs exhibit a significantly prolonged serum half-life when compared with other serum proteins.
Several key features of antibodies including but not limited to, specificity for target, ability to mediate immune effector mechanisms, and long half-life in serum, make antibodies and related immunoglobulin molecules powerful therapeutics. Numerous monoclonal antibodies are currently in development or are being used therapeutically for the treatment of a variety of conditions including cancer. Examples of these include Vitaxin™ (MedImmune), a humanized Integrin αvβ3 antibody (e.g., PCT publication WO 2003/075957), Herceptin® (Genentech), a humanized anti-Her2/neu antibody approved to treat breast cancer (e.g., U.S. Pat. No. 5,677,171), CNTO 95 (Centocor), a human Integrin αv antibody (PCT publication WO 02/12501), Rituxan™ (IDEC/Genentech/Roche), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma (e.g., U.S. Pat. No. 5,736,137) and Erbitux® (ImClone), a chimeric anti-EGFR antibody (e.g., U.S. Pat. No. 4,943,533).
There are a number of possible mechanisms by which antibodies destroy tumor cells, including anti-proliferation via blockage of needed growth pathways, intracellular signaling leading to apoptosis, enhanced down regulation and/or turnover of receptors, ADCC, CDC, and promotion of an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol., 11:541-547; Glennie et al., 2000, Immunol Today 21:403-410). However, despite widespread use, antibodies are not yet optimized for clinical use. Engineering IgGs for better binding to FcRn may represent a viable strategy for generation of therapeutic antibodies with increased serum persistence. Therapeutic antibodies that exhibited longer half-lives likely would be of benefit with increased efficacy because of sustained serum concentrations, decreased dosing frequency and/or lower cost of goods.
Various strategies have explored the effects of modulating the affinity of IgG molecules to FcRn on their serum persistence in vivo. In particular, several mutagenesis studies have targeted human Fc regions in an effort to decrease their binding affinity to human or murine FcRn at acidic pH. The serum half-lives of such engineered molecules were significantly reduced in mice expressing endogenous (Kim et al., 1999, Eur. J. Immunol., 29, 2819-2825) or human (Petkova et al., 2006, Int. Immunol., 18, 1759-1769) FcRn. Conversely, various Fc mutations have been described which resulted in significant increases in human or mouse IgG Fc binding to mouse (Ghetie et al., 1997, Nat. Biotechnol., 15, 637-640), rhesus monkey (Hinton et al., 2004, J. Biol. Chem., 279, 6213-6216; Hinton et al., 2005, J. Immunol., 176, 346-356) and cynomolgus monkey (Dall′ Acqua et al., 2006, J. Biol. Chem., 281, 23514-23524) FcRn. These mutated IgG molecules were reported to have significantly improved serum half-life in the corresponding hosts.
One particular set of mutations, M252Y/S254T/T256E (referred to as ‘YTE’), have been reported to result in an about 10-fold pH dependent increase in the binding of various humanized IgGs to both human and cynomolgus monkey FcRn at pH 6.0 (Dall′ Acqua et al., 2002, J. Immunol., 169, 5171-5180; Dall′ Acqua et al., 2006, J. Biol. Chem., 281, 23514-23524). When dosed in cynomolgus monkeys, the serum half-life of a YTE-modified humanized IgG was reported to be increased by nearly 4-fold when compared with its unmutated counterpart (Dall′ Acqua et al., 2006, J. Biol. Chem., 281, 23514-23524). The introduction of YTE into therapeutic IgGs could potentially provide many benefits such as reduced administration frequency and/or dosing requirements.
The three-dimensional structure coordinates of a crystalline Fc region with enhanced serum half-life, such as Fc/YTE, could enable one to elucidate a molecular mechanism of the enhanced interaction between Fc/YTE and FcRn. This three-dimentioanl structure coordinate could also be used to design and/or select Fc variants with altered (e.g., enhanced) FcRn binding affinity and serum half-life. Provided herein are the atomic structure coordinates of such Fc variants, particularly Fc/YTE.